Difference between revisions of "Part:BBa K1632010:Experience"
(→‎Materials and Methods)
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===Materials and Methods===
 
===Materials and Methods===
 
<b>1. Construction</b><br>
 
<b>1. Construction</b><br>
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All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br>
+
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
  
−
A. Ptet_RhlR (6A1)　Prhl-GFP (3K3) <br>
+
A. PBAD/araC_FimB(6A1) 　Fimswitch[default ON]_gfp(3K3) <br>
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B. Ptet_RhlR (6A1)　Prhl(RR)-GFP (3K3) <br>
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B. PBAD/araC_FimB(6A1) 　Fimswitch[default ON]_gfp(3K3) <br>
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C. Ptet_RhlR (6A1)　Prhl(LR)-GFP (3K3) <br>
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C. no gene(4A5) Fimswitch[ON]_gfp(3K3) …positive control <br>
−
D. Ptet_RhlR (6A1)　Prhl(RL)-GFP (3K3) <br>
+
D. no gene(4A5) Fimswitch[OFF]_gfp(3K3) …negative control <br>
−
E. Ptet_RhlR (6A1)　 Placuv5-GFP (3K3) …positive control<br>
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E. PBAD/araC_FimB(6A1) J23119_gfp(3K3) …positive control 2 <br>
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F. Ptet_RhlR (6A1)　promoter less-GFP(3K3) …negative control<br>
+
F. PBAD/araC_FimB(6A1)+rbs_gfp(3K3) …negative control 2 <br>
  
 
[[Image:Improved_Prhl_Promoter_Assay_Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
 
[[Image:Improved_Prhl_Promoter_Assay_Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>

Revision as of 11:27, 13 September 2015

Prhl(RL)-GFP

Materials and Methods

1. Construction
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.

A. PBAD/araC_FimB(6A1) 　Fimswitch[default ON]_gfp(3K3)
B. PBAD/araC_FimB(6A1) 　Fimswitch[default ON]_gfp(3K3)
C. no gene(4A5) Fimswitch[ON]_gfp(3K3) …positive control
D. no gene(4A5) Fimswitch[OFF]_gfp(3K3) …negative control
E. PBAD/araC_FimB(6A1) J23119_gfp(3K3) …positive control 2
F. PBAD/araC_FimB(6A1)+rbs_gfp(3K3) …negative control 2

Fig. 1. Plasmids

2. Assay protocol
1. Prepare overnight cultures for the each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL)　and glucose (final concentration of mass of glucose is 0.5 percent) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 0.5 percent).
3.Grow the cells at 37 ℃ until the observed OD590 reaches 0.4 (Fresh Culture)
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant by using P1000 pipette
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant by using P1000 pipette
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
9. Remove the supernatant by using P1000 pipette
10. Add 1 mL of LB containing Amp and Kan, and suspend.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan, glucose (final concentration of mass of glucose is 0.5 percent) and 30 microL of sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)
12. Grow the samples at 37 ℃ for 6.5 hours.
13. Measure OD590 of all the samples every hour.
14. Start preparing the flow cytometer 1 h before the end of incubation.
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
16. Remove the supernatant by using P1000 pipette
17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
18. Dispense all of each suspension into a disposable tube through a cell strainer.
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)